DNA sequencing is a rapidly developing field with key players such as Illumina (using the Solexa technology), Life (using the Solid technology) and Roche Diagnostics (using the 454 technology). The drawback of the sequencing methods these companies use is that the sequence information is obtained by a repetition of several steps which involve replacing reagents and washing. This is cumbersome and time consuming and wastes a lot of expensive reagents. The number of repetitions is equal to the number of nucleotides that are interrogated, i.e. the read length. With the desire to increase read length this problem will become more severe in the future. Currently, this is compensated by highly parallel sequencing on large arrays. However, for clinical applications this is less desirable. One would rather have a fast answer and a lower multiplexing. At the same time the cost per base pair has to come down significantly. Since after every step, i.e. after nucleotide incorporation, the whole surface needs to be washed, all reagents end up in the waste. The total reagent consumption is proportional to the read length and the dominant cost factor of sequencing at the moment.
The process of sequencing a particular target, such as e.g. DNA becomes more complex, since the incorporation reaction needs to be followed by an activation reaction and in between careful washing steps are required.
Alternative approaches, like that of Pacific Biosciences are more efficient as they follow the incorporation of nucleotides in real time for every molecule separately. In this way no washing is required. The optical requirements for such a system on the other hand are very severe, as one needs single fluorophore sensitivity for 4 different colors in real time. This can only be achieved for a limited area as the field of the objective lenses with high magnification is limited for practical systems and a strong laser light source. With the limited area only a small selection of a sample can be sequenced and the risk of errors due to missed reads is high. The fluorescent signals from labeled nucleotides while they are built in by the polymerase need to be discriminated from those of the same kind of molecules which happen to be at the same position by chance. This is done by analyzing the pulse length of the fluorescence signal.